The principal goal of this proposal is to develop new genetic tools that permit the rapid construction of repair-defective pathogenic bacteria. Our approach is called TAISR (for Trans-Acting Inhibition of SOS Repair) which introduces vectors carrying dominant mutant genes that "poison" SOS repair activities. A requirement for DNA repair functions in pathogenesis has been documented for some intracellular pathogens. For example, repair-defective mutants of Salmonella were completely attenuated in mice; this loss of virulence was traced to their inability to repair DNA damage within macrophages. A number of pathogens not only survive but even flourish within professional phagocytes; these include some Brucelleae, Campylobactereae, Edwardsielleae, Franciellaea, Listereae, Mycobacterieae and Yersineae. We are investigating the role of repair in pathogenesis by Brucella abortus an intracellular pathogen that causes undulant fever in humans and induces abortions in animals. We found that some B. abortus repair mutants, constructed by gene-disruptions, can decrease its ability to survive and grow in macrophages by 3,000-fold. Preliminary studies using a lexA-based TAISR system revealed partial poisoning of B. abortus SOS repair and a 100-fold greater killing in macrophages. We are proposing to improve TAISR by developing more effective dominant genes as well as use mutant combinations to inactivate repair in B. abortus. Attenuated mutants or mutant combinations generated by TAISR will provide insights into the design of live vaccines against Brucella. These genetic tools will have broad applications; they may be used to construct attenuated mutants in variety of bacterial pathogens. We are proposing the following specific aims for developing convenient genetic tools to construct attenuated mutants of Brucella. 1) Characterization of repair-defective B. abortus carrying dominant mutants from E. coli (lexA, recA and ruvB) as well as mutant combinations. 2.) Functional and molecular characterization of cloned copies of the lexA, recA and ruvB homologs from B. abortus. 3) Site-directed mutagenesis to construct dominant mutants from these three B. abortus repair genes. 4) Characterization of B. abortus strains expressing dominant lexA, recA and ruvB mutants from B. abortus.